Inherited deficiency of human lysosomal proteins (including hydrolases, transport proteins, receptor molecules, ion pumps, and small “activator” molecules) leads to lysosomal storage disorders or LSD (Hers, 1963, Biochem. J. 86: 11-16; Scriver et al., 2000, The Metabolic and Molecular Bases of Inherited Disease, 8th ed., McGraw-Hill), one of the most prevalent genetic defects, affecting one out of seven thousand live births (Meikle et al., 1999, JAMA 281: 249-254). More than 40 forms of LSD have been identified in humans. Their manifestations include accumulation of non-graded substrates, lysosomal engorgement, cell damage and tissue dysfunction (e.g., neuropathy, pulmonary, renal, and hepatic disorders), increased morbidity, and premature mortality (Reviewed by Scriver et al., 2000, The Metabolic and Molecular Bases of Inherited Disease, 8th ed., McGraw-Hill).
Current therapies, limited to symptomatic palliative treatment, only very marginally improve LSD prognosis (Scriver et al., 2000, The Metabolic and Molecular Bases of Inherited Disease, 8th ed., McGraw-Hill). New approaches are focused on gene therapy and transplantation of lysosomal protein-secreting cells or bone marrow stem cells (Malatack et al., 2003, Pediatr. Neurol. 29: 391-403; Krivit et al., 1992, Bone Marrow Transplant 10 (Suppl. 1): 87-96; Haskins, 1996, Bone Marrow Transplant. 18 (Suppl. 3): S25-S27; Jin et al., 2003, Mol. Ther. 8: 876-885; Miranda et al., 2000, Gene Ther. 7: 1768-1776; Miranda et al., 1998, Transplantation 65: 884-892; Leimig et al., 2002, Blood 99: 3169-3178; Hoogerbrugge et al., 1988, J. Clin. Invest 81: 1790-1794; Yeyati et al., 1995, Hum. Gene Ther. 6: 975-983). However, practicality of gene therapies is limited by abnormal processing of newly synthesized lysosomal proteins achieved by gene transfection and general concerns on safety and effectiveness of this strategy (D'Azzo, 2003, Acta Haematol. 110: 71-85; Cabrera-Salazar et al., 2002, Curr. Opin. Mol. Ther. 4: 349-358; Taylor et al., 1997, Nature Med. 3: 771-774). Lack of compatible donors, risk of body irradiation and immunosuppressive agents and graft-versus-host diseases hinder applicability of transplantation (Estruch et al., 2001, J. Gene Med. 3: 488-497; Miranda et al., 1997, Blood 90: 444-452; Vellodi et al., 1997, Arch. Dis. Child. 76: 92-99; Miranda et al., 2000, Faseb J. 14: 1988-1995; Jin et al., 2002, J. Clin. Invest 109: 1183-1191).
At the present time, protein replacement therapy seems to be the most feasible treatment, in particular in the case of non-neurological LSD (e.g., Gaucher I, Fabry disease, Niemann-Pick B (Reviewed by Brady, 2003, Phil. Trans. R. Soc. London B Biol. Sci. 358: 915-919; Grabowski and Hopkin, 2003, Annu. Rev. Genomics Hum. Genet. 4: 403-436; Desnick and Schuchman, 2002, Nature Rev. Genet 3: 954-966; Barton et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1913-1916; LeBowitz et al., 2004, Proc. Natl. Acad. Sci. USA 101: 3083-3088). The principle of LSD therapy by infusion of exogenous proteins is based on the phenomenon that endogenous human lysosomal proteins (e.g., enzymes such as hydrolases that needed to be replaced) are normally modified with mannose and/or mannose-6-phosphate (M6P) residues and, therefore, can bind to cellular mannose or M6P receptors (MR, M6PR), followed by internalization via clathrin-mediated pathways and rapid trafficking to lysosomes (Kaplan et al., 1977, Proc. Natl. Acad. Sci. USA 74: 2026-2030; Kornfeld et al., 1982, CIBA Found. Symp. (92): 138-156).
Given these specific glycosylation requirements, production of recombinant proteins for the treatment of LSD cannot take advantage of typical expression systems (e.g., bacteria, yeast, or insect cells), but these proteins must be produced in mammalian cells (Boose et al., 1991, Glycobiology 1: 295-305). Nevertheless, even when produced in mammalian systems (e.g., CHO cells), some lysosomal proteins are poorly or differently modified (Waheed et al., 1988, EMBO J. 7: 2351-2358; He et al., 1999, Biochim. Biophys. Acta 1432: 251-264; Miranda et al., 2000, FASEB J. 14: 1988-1995; Zhu et al., 2004, J. Pharmacol. Exp. Ther. 308: 705-711), thus lack of mannose residues or accessibility of these for receptor binding are common obstacles affecting the targeting capacity of recombinant lysosomal proteins (Murray, 1987, Meth. Enzymol. 149: 25-42).
Strategies to overcome this problem focused on increasing effective exposure of mannose residues by in vitro modification of the lysosomal proteins (e.g., digestion of oligosaccharide side chains) (Murray, 1987, Meth. Enzymol. 149: 25-42; Furbish et al., 1981, Biochim. Biophys. Acta 673: 425-434; Brady et al., 1994, J. Inherit. Metab. Dis. 17: 510-519). Moreover, augmentation of the dose of the infused proteins has been utilized to compensate for their relative targeting inefficiency, but this strategy will likely result in detrimental side effects (e.g., immunogenic responses), together with rapid saturation of the receptor-mediated uptake (Mistry et al., 1996, Lancet 348: 1555-1559; Friedman et al., 1999, Blood 93: 2807-2816). Alternative means to enhance the efficiency of enzyme replacement therapy for LSD included administration of dexamethasone, to increase the density of surface MR (Zhu et al., 2004, J. Pharmacol. Exp. Ther. 308: 705-711). However, protein delivery to MR, preferentially expressed in the reticulo-endothelial system (RES), precludes other cells and tissues from being efficiently targeted (Stahl et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1399-1403; Achord et al., 1978, Cell 15: 269-278).
Related receptors used by endogenous lysosomal proteins, namely M6PR (cation-independent and cation-dependent receptors), can be utilized to deliver the infused recombinant proteins to cell types other than RES (Kornfeld, 1990, Biochem. Soc. Trans. 18: 367-374; Kornfeld, 1987, FASEB J. 1: 462-468; Sands et al., 2001, J. Biol. Chem. 276: 43160-43165). Nevertheless, also lack of M6P residues in recombinant lysosomal proteins leads to rapid clearance of these from the circulation (He et al., 1999, Biochim. Biophys. Acta 1432: 251-264). To circumvent this obstacle, a recent work focused on targeting of a recombinant lysosomal protein (e.g., β-glucoronidase) in a glycosylation-independent manner, by generating a fusion protein tagged with a peptide derived from the insulin-like growth factor II (IGF II), which acts per se as a ligand for M6PR (LeBowitz et al., 2004, Proc. Natl. Acad. Sci. USA 101: 3083-3088). However, in contrast to control cells, recent evidence showed defective internalization via clathrin-mediated pathways in LSD cells, which may also hinder this strategy (Dhami et al., 2004, J. Biol. Chem. 279: 1526-1532; Prince et al., 2004, J Biol Chem. 279(33): 35037-46).
To circumvent this obstacle, two recent works focused on targeting of recombinant lysosomal enzymes (β-glucoronidase, alpha-L-iduronidase and acid alpha-glucosidase) in a glycosylation-independent manner, by generating fusion proteins tagged with: i) a peptide derived from the insulin-like growth factor II (IGF II), which acts per se as a ligand for M6PR (LeBowitz et al., 2004, Proc. Natl. Acad. Sci. USA 101: 3083-3088); or ii) a peptide derived from receptor associated protein, RAP, which binds to several LDL receptor family members (Prince et al., 2004, J Biol Chem. 279(33): 35037-46). However, both these targets are internalized within cells by clathrin-mediated pathways, which are defective in LSD cells, also hindering the efficacy of this strategy (Dhami et al., 2004, J. Biol. Chem. 279: 1526-1532; Section 6.4 below).
Therefore, effective intracellular delivery of injected lysosomal proteins to their ultimate target, lysosomes, has not been achieved by known formulations and methods for enzyme replacement therapy.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.